Cell line expressing mutated human tissue-type plasminogen activator, the constructing strategy thereof and method of preparing expressed protein

ABSTRACT

The invention involves a cell line which can express human mutated tissue-type plasminogen activator (TNK-TPA), and its preparation methods. The collection No. of cell line in this invention is CCTCC C200006. We firstly use a DNA fragment from the short arms of human D, G group chromosomes or its homologous to construct a recombinant human source gene vector-TNK-TPA (collection number is CCTC m200032); TNK-TPA gene is then transferred into the target site of nucleolus organizing region (NOR) of human D, G group chromosomes in host cell HT1080 by the recombinant and the cell line is attained after screening, which can be used for manufacturing protein.

SUMMARY OF THE INVENTION

[0001] The invention involves a cell line specifically expressingmutated human tissue-type plasminogen activator (TNK-TPA), cell line'sconstruction strategy and its application in manufacturing recombinantgene medicine of human tissue-type plasminogen activator.

BACKGROUND OF THE INVENTION

[0002] Human tissue-type plasminogen activator(t-PA) can dissolve localthrombus in blood vessel and recanalize the blocked vessel by activatingplasminogen. At present, American Genetech Company is the only one candevelop recombinant human t-PA(rt-PA) at a large scale by means of geneproject technology and has developed successfully rt-PA medicine. Thefirst generation of rt-PA made in this company has become of choicetreatment to thrombotic diseases, but it has some shortcomings. First,it's price is rather high with $1375 per vial on international market;second, its function has some weak points: 1)short half-life, just 3 to5 minutes; 2)low specific affinity with fibrin of gore; 3) activity andefficacy should be further improved. Much more medicine dose should begiven because of short half-life. Low specificity and activity give riseto two results: on one hand, there is still 10-20% patient's thrombuscan't be completely dissolved by rt-PA, on the other hand, it canresults in fibrinolysis over all the body and affects normal cruormechanism so as to cause severe complications such as bleeding,especially in skull and digestive tract. In terms of the above reasons,some foreign companies have got down to develop the second generation ofrt-PA since the -PA was used in the clinical. They modified the rt-PAgene in order to eliminate or weaken the above weak points. TNK-TPA is akind of mutated form which is universally researched. For example, KeytB A, Paoni N F, Refino rt C J, et al proclaimed “a Faster-acting AndMore Potent Form of Tissue Plasminogen Activator”. ((Proc Natl Acad SciUSA) Apr. 26, 1997; 91(9):3670-4.). It was constructed on the basis ofthree sites of t-PA gene site-directed mutation. Two sites locate inKringlel function domain, T103N and Q117N; one locates in Proteasedomain, KHRR296-299AAAA.T103N adds a glycosylation site which increasesspecificity of combination of TNK-tPA and fibrin and decreases itscleanup rate; Q117N destroys combination of K1 region and mannose andthe action domain of KUPFFER cell in liver, and prolongs half-life;KHRR296-299AAAA destroys functional region of PAI-1 and prolongs itshalf-life, too. As for the development of TNK-TPA, it is universal toadopt ordinary eukaryotic gene expression vector, using CHO cell as hostcell, TNK-TPA integrate randomly into the expression system of host cellgenome. For example, Wu Benchuan, et al announced the above expressionsystem and preparation method of expressed protein in the article“Purification and identification of recombinant tissue-type plasminogenactivator” published in <Development of Biochemistry andBiophysics>(1997,24 (1):71-75)

DETAILED DESCRIPTION OF THE INVENTION

[0003] The inventor discovered two families carrying an extrabi-satellite microchromosome (BM) in these two families with 2 and 3generations, the BM does no harm to human body. The families show normalphenotype. It is reported 17 similar families in the world, and nobodyconceived of assembling human gene vector using the small chromosomeelements. The applicant put forward the structure. He found out that thechromosomes come from short arms of human D, G group chromosomes 13, 14,15, 21, 22 by FISH technique. The short arms of human D, G group arenucleolus organizing region abundant in ribosome DNA (rDNA) . Theseregions exist different polymorphisms with different length in human,and the genes in these regions can be actively transcribed in the cellmetaphase. So he inferred that specific DNA fragments can be used asleading sequence if they were isolated from BM, and the gene of interestcan be transferred into nucleolus organizing region in short arms ofhuman D, G group chromosomes, the gene should be relatively highly,stably and harmlessly expressed. The following example can prove thisstrongly.

[0004] The inventor at first constructed specific pUC19 library of themicrochromosomes by micro-dissection method and got single copy fragmentby screening the library. The fragment proved by FISH technique comesfrom short arms of human D, G group chromosomes and the single copy wasused as probe to screen human PAC genomic library. Then, a 120 kb of BMspecific DNA fragment which comes from BM and short arms of human D, Ggroup chromosomes was confirmed by FISH technique (FIG. 1). Geneconcerned important physiological function is not found by analyzingsequence of this BM specific fragment (BMSF). So it is safe to take itas target. The applicant further used a small fragment from BMSF asleading sequence to construct gene vector.

[0005] Based on above evidences, the applicant holds that a gene vectorby using the DNA sequence without vital physiological function-relatedgene or a DNA sequence with identity to short arms of human D, G groupchromosomes as the leading sequence can site-directly introduce targetgene to the short arms of human D, G group chromosomes and the cell linefor this invention can then be obtained.

[0006] Different vectors can be constructed on the basis of leadingsequence according to the present technique, and then we can getrecombinant vector-TNK-TPA by ligating TNK-TPA gene into the constructedvector. The construction methods of vector and recombinantvector-TNK-TPA are commonly used. Using the recombinant above, thetarget gene TNK-TPA can be also transferred into host cell by routinemethod.

[0007] The example of the invention gives in detail the identificationof the 120 kb specific DNA sequence from short arms of human D, G groupchromosomes, the construction of the vector using a 3.8 kb fragment fromthe 120 kb BMSF as leading sequence. The full sequence of the vector isgiven in SEQ No 1. Then target gene is ligated into the vector andrecombinant vector-TNK-TPA is achieved. The inserting site of TNK-TPAgene is at 5910 of the vector. The bacterial strain containedrecombinant vector-TNK-TPA is preserved in the China Typical CultureCollection Center (Wuhan University, China. Wuhan 430072) on Sept. 29,2000, collection number is CCTCCM200032. The preservator names itEscherichia coli JM109/JH-5/pNS-TPA.

[0008] According to the way above, the invention got a novel cell linewith target gene TNK-TPA integrated in short arms of D, G groupchromosomes of HT1080 cell. The cell line was accepted by China TypicalCulture Collection Center (Wuhan University, China. Wuhan 430072) onAug. 18, 2000, and preservation number is CCTCC C200006. The cell lineis human fibrosarcoma cell line, which is human gene-transferred cell.The preservator names it human fibrosarcoma cell line/JH-2/TPA. Thepreserved is a pure cultivative cell line, the culture condition is 10%calf serum in DMEM, PH is 7.0-7.4, 37° C.

[0009] The above cell line can substitute CHO transformed form toprepare TNK-TPA.

[0010] The purified protein expressed by the cell line of the inventionhas been confirmed to be the target protein by Western blotting andamino acid sequence analysis.

[0011] The invention obtained a novel cell line by transferringsite-directly recombinant human-source gene vector/TNK-TPA cDNA into D,Ggroup chromosomes of human HT1080 cell and gave a method to produceTNK-TPA. Compared with the existing technology, it dramaticallydecreases cost of TNK-TPA because of its efficient and stableexpression.

[0012]FIG. 1 shows FISH mapping of 120 kb fragment cloned in PAC;

[0013]FIG. 2 is a circular map of the gene vector described in theinvention (length of gene vector sequence: 11162 bp); pGEM-7(8267-11162):vector replication component and prokaryote screeningsystem; TK(1-2840): eukaryote negative screen gene, using TK promoterand TK polyA signal; Neo(4342-5910):eukaryote positive screen gene,using sv40 promoter and sv40 polyA signal; GLS(2841-4341,5911-8267):leading sequence for gene targeting; Cloning site(5910): insert-site of target gene;

[0014]FIG. 3 is FISH mapping of targeted TNK-TPA gene in positivelytransformed cell colony, this revealed that TNK-TPA gene has beensite-directly transferred into short arms of D,G group chromosomes bythe vector;

[0015]FIG. 4 is Western blotting result of purified TNK-TPA protein, 1˜4are purified TNK-TPA protein, “−” suggests negative control;

EXAMPLE ONE

[0016] Preparation of Gene Leading Sequence in the Invention

[0017] 1 Isolation of PAC Clone With Gene Leading Sequence

[0018] 1.1 Construct BM Specific pUC19 Library through Micro-Dissection,PCR, Microcloning (Deng H-X, Yoshirua K, Dirks R W, et al. Hum Genet1992,89:13.)

[0019] 1.2 Identification of BM Specific Single Copy DNA

[0020] (1) preparation of colony matrix membrane: draw a 14×14 squarewells on two nylon membranes, labeled as A and B, and put into twoplates with solid LB, respectively, then pick white clone randomly fromlibrary plates and inoculate to the 14×12 square wells of two membraneswith same coordinate, add 100 ng single copy DNA to the 13th line aspositive control, and then add 100 ng gDNA to 14th line as negativecontrol, two plates are incubated for 10˜12 hours at 37° C., andmembrane B is stored at 4° C., remove membrane A from plate, and treaton a filter paper soaked by following solution: 10% SDS, 5 minutes, 0.5NNaOH/1.5M NaCl, 3 minutes, 1.5M NaCl/0.5M Tris.HCl, 3 minutes,2×SSC/0.2M Tris.HCl, 10 minutes, 80° C. vacuum dry 2 hours, keep foruse.

[0021] (2) Preparation of gDNA Probe

[0022] Sample 50˜75 ng gDNA, add asepsis water to 11 ml, boil anddenature at 100° C. for 10 minutes, the following system is used asrandom primer labeling reaction: 2 mM dNTP(dATP⁻) 3 μl primer mixture 2μl Klenow enzyme 1 μl α-³²p-dATP 3 μl

[0023] vortex and mix up thoroughly, incubate 30 minutes at 37° C. Add 8μl stop mixture, pass through G-50 column to purify probe, withdraw{fraction (1/10)} of the mixture to conduct liquid scintillation.

[0024] (3) Hybridization: colony matrix membrane is immersed in 2×SSC 10minutes, wipe out lightly colony fragments on membrane surface, 65° C.,pre-hybridize in 5 ml hybridization solution over 30 minutes. Sampleprobe solution to boil and denature 10 minutes at 100° C. according tothe concentration of 1.2×10⁶ cpm/ml and measure liquid scintillationvalue, add 5 ml fresh hybridization solution , hybridize with colonylattice membrane over 12 hours at 65° C.,wash membrane according tofollowing conditions: 2×SSC/0.1%SDS, 10 minutes at room temperature,2×SSC/0.1% SDS, 10 minutes at 65° C., 0.1%×SSC/0.1% SDS, 10 minutes at65° C., −70° C., autoradiograph, none or weak hybridization signal isprimarily regarded as single copy.

[0025] (4) Sequence analysis, Southern blotting: pick clone withouthybridization signal on the corresponding position of membrane B,culture it in small scale, sequence plasmid DNA extracted, the sequenceof single copy DNA shows no similarity compared with the database ofGenBank. At last, digest the plasmid by EcoR I enzyme and isolate theinsert DNA by random primer method, label the insert by α-³²p-dATP andhybridize with gDNA digested by EcoRI on a piece of nylon membrane,those showing 1 or 2 hybridization bands are single copy DNA clones.

[0026] 1.3 Identification of PAC Clone with BM Specific Sequence andShort Arms of Human D, G Group Chromosomes

[0027] (1) Screen Human PAC gDNA and Acquire Positive Clone

[0028] (2) Label 260 bp single copy probe P8-7 with α-³²p-dATP by randomprimer method→purify probe by G-50 column (medium granularity)→ready foruse 4° C.→immerse 7 PAC membranes in 2×SSC 10 minutes→pre-hybridize 3hours, 55° C.→denature probe 10 minutes, 100° C.→add to 50 mlhybridization solution purchased commercially to a final concentrationof 4.6×10⁵ cpm/ml and hybridize with PAC membrane 1 hour 65° C.→washmembrane: 2×SSC , once a time 10 minutes at room temperature, 2×SSC/0.1%SDS, 10 minutes at 65° C. twice→apply to X-ray film, autoradiograph at70° C. 12 hours→develop X-ray film→read the positive clones numberfollowing instruction.

[0029] (2) Pick randomly positive clones and number from five differentplates, purchase PAC clone.

[0030] 1.4 Hybridization of the DNA fragment in PAC clone with metaphasecell confirm that the insert DNA in positive PAC clone originates fromshort arms of human D, G group chromosomes through FISH, as the FIG. 1shows.

[0031] The above experimental methods refer to the Molecular Cloningwritten by J Sambrook et al., second Edition, Cold Spring Harborlaboratory Press, 1989.

[0032] 2. Isolation of the Leading Sequence.

[0033] Main material: βagarase(Bio-Labs), Not I Agarose

[0034] (1) Digest the plasmid PAC169 by Not I enzyme

[0035] (2) Isolate an around 120 kb insert DNA through PFGE. Pulseelectrophoresis conditions: electrophoresis buffer: 0.5×TBE Highstrength Analytical Grade Agarose: (Bio-Rad, low Melting point agaroseLMP) 1%, Switch time: from two to fifteen second, electrophoresis time:18 h. voltage: 6 v/cm, angle: 120°, temperature: 14° C.

[0036] (3) After electrophoresis, stain the gel with EB (0.2 ug/ml)thirty minutes. According to the Marker display, cut that 120 kb DNAband with sterile knife.

[0037] (4) Treat the gel cut out by β-agarase and precipitate withenthanol.

Example 2

[0038] Preparation of Gene Vector in the Invention:

[0039] 1. The Construction of Gene Vector and Induction of Target Gene

[0040] 1.1 Construct Gene Vector

[0041] 1.1.1 Nsi I and Stu I (blunt enzyme) digest PAC DNA, run thesample on a common agarose gel, recover the 3.8 kb DNA and purify theDNA by electric elution.

[0042] 1.1.2 pGEM-TK vector DNA is digested with Hind III, and thendigested with Klenow further to generate the blunt ends.

[0043] 1.1.3 The blunt ends of pGEM-TK/Hind III is further digested withNsi I.

[0044] 1.1.4 The purified 3.8 kb/Nsi I+Stu I digested DNA and the Nsi Idigested pGEM-TK are ligated for 17 hours at 16° C.

[0045] 1.1.5 The ligated product is transformed into JM109 competentbacteria, culture 18 hours at 37° C. in an ampicillin plate.

[0046] 1.1.6 Pick randomly monoclones and identify the positive cloneswith Nsi I and Nhe I double digestion.

[0047] 1.1.7 The plasmid pCDN-GPR is digested with Xba I and Nhe I toacquire the Xbal+Nhel digested Neo gene.

[0048] 1.1.8 Construct pNS2 gene vector by ligating the Xbal+Nheldigested Neo gene with the pGEM-TK-3.8 kb/Nhe I fragment.

[0049] 2.1 Induction of TNK-TPA Gene

[0050] 2.1.1 Clone TNK-TPA (CDS) into pcDNA3.1(−).

[0051] 2.1.2 Design the primers TPCF and TPCR to amplify TNK-TPA geneand expression element (CMV promoter and BGH polyA signal), Avr IIrestrictive sites are added to the two ends of the primers. PrimerSequence: TPCF: ATGCATCCTAGGGGAGGTCGCTGAGTAGTG        AvrII TPCR:TGCATGCCTAGGTACCCCCTAGAGCCCAG        AvrII

[0052] 2.1.3 Digest TNK-TPA gene and expression element (CMV promoterand BGH polyA signal) with AvrII, ligate it to the pNS2 vector digestedwith Nhe I.

[0053] 2.1.4 Transform the ligated product into recombinant JM 109 E.coci and gain bacterial strain containing vector-TNK-TPA (collectionnumber is CCTCC M200032).

[0054] The above experimental methods refer to the Molecular Cloningwritten by .J Sambrook et al., second edition, Cold Spring Harborlaboratory Press. 1989.

[0055] 3 The Gene Vector Extraction.

[0056] 3.1 Material:

[0057] 3.1.1 QIAGE Plasmid Maxi Kit

[0058] 3.1.2 Culture Medium: Liquid LB Trypton   5 g Yeast extract 2.5 gNaCl 2.5 g Add H₂O to 500 ml Sterilize by autoclaving

[0059] 3.1.3 Ampicillin: 100 mg/ml(1000×)

[0060] 3.2 Method:

[0061] 1) Pick positive monoclones and inoculate them in 3 ml LB (Amp+),incubate for 1 h at 37° C., 250 rpm .

[0062] 2) Inoculate 100 ul of primary culture in 100 ml LB (Amp+),incubate for 16 h at 37° C., 250 rpm.

[0063] 3) Harvest the bacteria by centrifugation at 6000×g for 15minutes at 4° C.

[0064] 4) Resuspend the bacterial pellet in 10 ml of buffer P1.

[0065] 5) Add 10 ml of buffer P2, mix gently but thoroughly 6 times, andincubate at room temperature for 5 minutes.

[0066] 6) Add 10 ml of chilled buffer P3, mix gently by inverting 6times, and incubate on ice for 20 minutes.

[0067] 7) Centrifuge at 2000×g for 30 min at 4° C.

[0068] 8) Transfer supernatant containing plasmid DNA promptly into a 40ml high-speed centrifuge tube and centrifuge at 2000×g for 15 min at 4°C.

[0069] 9) Equilibrate a QIGEN tip 500 by applying 10 ml buffer QBT.

[0070] 10) Transfer the supernatant to the QIGAE-tip 500 and allow it topass through this column.

[0071] 11) Wash the QIGAE-tip with 30 ml buffer QC.

[0072] 12) Elute DNA with 15 ml QF and collect elution.

[0073] 13) Precipitate DNA by adding 10.7 ml (0.7 volumes)isopropylalcohol to the elution, mix up.

[0074] 14) Centrifuge immediately at 15000×g for 30 min at 4° C.

[0075] 15) Remove the supernatant, wash DNA pellet with 5 ml of 70%ethanol and centrifuge at 15000×g for 10 min.

[0076] 16) Remove the 70% ethanol, air-dry the pellet for 10 min, anddissolve the DNA in TE solution.

Example 3

[0077] Transfer the recombinant vector-TNK-TPK acquired in the example 2into host cell HT1080, obtain the required cell lines after screening,and express in vitro, confirms site-directed integration and highefficient expression.

[0078] 1 Material:

[0079] 1.1 Cell: HT1080. Culture medium: high sugar DMEM+10% FBS(HT1080)EMEM+10% FBS.

[0080] 1.2 Electroporation Apparatus: Purchased from Bio-Rad Company.

[0081] 2.Methods

[0082] 1) Culture the cells in a 75 cm² culture bottle to 70%˜80%confluence.

[0083] 2) Harvest the cells and wash with HeBs Buffer for two times,then account the cells.

[0084] 3) Centrifuge at 15000 rpm at 4° C. for 10 min.

[0085] 4) Resuspend the cells with a reasonable volume of HeBS Buffer tomake the cell density to 10⁶˜10⁷ ml.

[0086] 5) Add 0.8 ml suspension (about 10 μg DNA vector) to an 0.4 cmelectroporation cuvette

[0087] 6) Electroporate the suspension, parameters: 260V, 550 uF for11˜13 ms.

[0088] 7) Transfer the shocked cells to a 75 cm² culture flask, add 14ml culture medium with ampicillin/streptomycin, then culture in 37° C.,5% CO₂ for 24˜48 hrs.

[0089] 8) Screen the cell with G418 (final concentration of 300 μg/ml),change medium once every 2-3 days, in parallel, normal cells is used ascontrol.

[0090] 9) Calculate the survival clone number in transfected cell afternormal cells completely die in 7-10 days, and then add G418 (150 μg/ml)to maintain it.

[0091] 10) Continue to screen the transfected cells with GCV (theultimate concentration of 500 ng/ml)

[0092] 11) Most cells die in 7-10 days, add GCV 250 μg/ml to remainliving cells or withdraw GCV, culture the cell up to 70%˜80% confluence,and determine its expressing activity of the transfected gene.

[0093] 3. Results

[0094] The TNK-TPA gene with the vector were transferred into HT1080cells by electroporation and the positive cell lines were obtained afterpositive and negative screening (Collection NO. CCTCC: C200006), andconfirmed to be site-directed insertion by FISH (FIG. 3) .The results ofactivity assay of TNK-TPA refer to Table 1.

[0095] Expression activities are: negative control HT1080 cells is 0U/10⁶ cells/ 24 hrs, the positive HT1080 cells is 408 U/10⁶/24 hrs aftertransfer and 407 U/10⁶ cells/24 hrs after 95 days. The expression ofTNK-TPA is of highly stability. In addition, expressed protein ofTNK-TPA is proved by Western Blotting and amino acid sequencing (FIG.4). TABLE 1 The results of activity assay of TNK-TPA for positive HT1080cells(ug/10⁶ cells/24 hrs) The days The days of transfer T1* of transferT15* 33 408 54 188 37 396 58 204 60 411 95 114 68 430 74 430 88 441 90440.9 95 407

[0096]

1 3 1 11162 DNA Homo sapiens misc_feature (1)..(11162) target genevector 1 gggcgaattg ggcccgacgt cgcatgctcc tctagactcg aggaattctaccgggtaggg 60 gaggcgcttt tcccaaggca gtctggagca tgcgcttaag cagccccgctgggcacttgg 120 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtaggcgccaaccg 180 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagtcaggaagttc 240 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagcacgtctcact 300 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcctttggggcagc 360 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaaggggtgggtcc 420 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtcctccggaggc 480 ccggcattct gacgcttcaa aagcgcacgt ctgccgcgct gttctcctcttcctcatctc 540 cggcctttcg acctgcagcg acccgcttaa cagcgtcaac agcgtgccgcagatcttggt 600 ggcgtgaaac tcccgcacct cttcggcaag cgccttgtag aagcgcgtatggcttcgtac 660 ccctgccatc aacacgcgtc tgcgttcgac caggctgcgc gttctcgcggccatagcaac 720 cgacgtacgg cgttgcgccc tcgccggcag caagaagcca cggaagtccgcctggagcag 780 aaaatgccca cgctactgcg ggtttatata gacggtcctc acgggatggggaaaaccacc 840 accacgcaac tgctggtggc cctgggttcg cgcgacgata tcgtctacgtacccgagccc 900 gatgacttac tggcaggtgc tgggggcttc cgagacaatc gcgaacatctacaccacaca 960 acaccgcctc gaccagggtg agatatcggc cggggacgcg gcggtggtaatgacaagcgc 1020 ccagataaca atgggcatgc cttatgccgt gaccgacgcc gttctggctcctcatatcgg 1080 gggggaggct gggagctcac atgccccgcc cccggccctc accctcatcttcgaccgcca 1140 tcccatcgcc gccctcctgt gctacccggc cgcgcgatac cttatgggcagcatgacccc 1200 ccaggccgtg ctggcgttcg tggccctcat cccgccgacc ttgcccggcacaaacatcgt 1260 gttgggggcc cttccggagg acagacacat cgaccgcctg gccaaacgccagcgccccgg 1320 cgagcggctt gacctggcta tgctggccgc gattcgccgc gtttacgggctgcttgccaa 1380 tacggtgcgg tatctgcagg gcggcgggtc gtggcgggag gattggggacagctttcggg 1440 gacggccgtg cccgccccag ggtgccgagc cccagagcaa cgcgggcccacgaccccata 1500 tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggcccccaacggcgacc 1560 tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgtcccatgcacg 1620 tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctgctgcaactta 1680 cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacgatctgcgacc 1740 tggcgcgcac gtttgcccgg gagatggggg aggctaactg aaacacggaaggagacaata 1800 ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgcacgggtgttgg 1860 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcgataccccacc 1920 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccaccccaccccccaag 1980 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaagccctgccatagccac 2040 gggccccgtg ggttagggac ggggtccccc atggggaatg gtttatggttcgtgggggtt 2100 attattttgg gcgttgcgtg gggtcaggtc cacgactgga ctgagcagacagacccatgg 2160 tttttggatg gcctgggcat ggaccgcatg tactggcgcg acacgaacaccgggcgtctg 2220 tggctgccaa acacccccga cccccaaaaa ccaccgcgcg gatttctggcgccgccggac 2280 gaactaaacc tgactacggc atctctgccc cttcttcgct ggtacgaggagcgcttttgt 2340 tttgtattgg tcaccacggc cgagtttccg cgggaccccg gccaggacctgcagaaattg 2400 atgatctatt aaacaataaa gatgtccact aaaatggaag tttttcctgtcatactttgt 2460 taagaagggt gagaacagag tacctacatt ttgaatggaa ggattggagctacgggggtg 2520 ggggtggggt gggattagat aaatgcctgc tctttactga aggctctttactattgcttt 2580 atgataatgt ttcatagttg gatatcataa tttaaacaag caaaaccaaattaagggcca 2640 gctcattcct cccactcatg atctatagat ctatagatct ctcgtgggatcattgttttt 2700 ctcttgattc ccactttgtg gttctaagta ctgtggtttc caaatgtgtcagtttcatag 2760 cctgaagaac gagatcagca gcctctgttc cacatacact tcattctcagtattgttttg 2820 ccaagttcta attccatcag aagctcctta attttatacc actgacttattttgaaggct 2880 gctataagaa acagccctat gaaactggta ttttcctact gcaaggtggctactttaaga 2940 caatttttca ttgcattcta tcaagggatg tcttattatt atatcattatatcaagtgat 3000 gttataaata gtaagaatca gattaagggc tcatatgtcc ttctttgtattgactgttga 3060 aaaggtatgg ggccaaattt gtagtttgtc tggaattaca tatttttgggggtctctatt 3120 atcttcatac ttatcctatc taaattttcc attgccaaat ttccttacttatttttagtt 3180 ttatcctatt gctcatgtat ttttatgtct ccataagtct attttggaaaaaggcagagt 3240 actcataatt ttagtatatc ttttagcttt atgttgccat aaacctttcattatatacat 3300 gatcaacaac agcaaattat ctcacttcag tatttagttt attattttacaaactgattt 3360 atgattgcta acatgtaact gaaggtatac actattagaa cacagttttcagtagaaagt 3420 agcactgcca ttgagtaaaa aaatgttcta acattagagc aacattcttatacaagtttg 3480 catgttgttt actgaggtct aaagcatgac tacacaaaag gctgaataaaattcagattc 3540 ttacatacac ataaaattgt tttattgaga tgacaaagta tatttattatgccacccaga 3600 atataatcca ctctgataac tgccagtgta tgcacttgct gaagtaactcagtacataaa 3660 tggtagccac aacagttgct gtgcatgaaa gttcttctct tccagattgaagagtgtaca 3720 atctaaagca ttttaaaact ttaaatccct tattagctta aatataatttaaaattttag 3780 tttgccgtac ctataatttg tctgtacact aggttactaa gggtgatatgattacatatg 3840 tggatacaaa ataattttaa tggaaaatga aattagggta ctcaacaaagataaagggta 3900 atgatcatgt acactaaccg tatttgagat tagtttaagc ctggggtagctatacttatg 3960 tttcacagac cttgagaaga tagggaaaaa aagcttttat caacattgctaaggaacagg 4020 taaaagctaa cattaggtaa ctaagaggtg acataaaaaa gactgaataaaatatcatgg 4080 aggtttcata ataagattgg aaattccata gactaggaga gaaaagatcccaaaatatac 4140 atgctcattg ggaaaacagc tagtaagaac aaggagagat ctctatttaatgatacaata 4200 gtagagttat aatttcctgt atattgtaaa tttcaagcat ttaaacattttcattgaatt 4260 ataaaatatt atttgtaaaa gaaagaaaaa cagcacaact gcagattacagatgactaag 4320 atagatgaat catgaaaagg tgctagattg tgagcggata acaatttcacacaggaaaca 4380 gctatgacca tgattacgcc aagctctcga cgggatcgcg gccgcgatccagacatgata 4440 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaaatgctttatt 4500 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaataaacaagtt 4560 ggggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatccagccggcgtc 4620 ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaatcgaaatctcg 4680 tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcccgctcagaag 4740 aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggcgataccgtaa 4800 agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatcacgggtagcc 4860 aacactatgt cctgatagcg gtccgccaca cccagccggc cacagtcgatgaatccagaa 4920 aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggtcacgacgaga 4980 tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctggcgcgagcccc 5040 tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccgagtacgtgct 5100 cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatcaagcgtatgc 5160 agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaaggtgagatgac 5220 aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgcttcagtgaca 5280 acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatagccgcgctgcc 5340 tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaagaaccgggcgc 5400 ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctgttgtgcccag 5460 tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaatccatcttgt 5520 tcaaccatgg tggatcgatc caagctccca acacaactat gtcagaagcaaatgtgagga 5580 gcaactgatc ctacctcacc ttatatgctc tgccctggct cctgccctctctatcctgtg 5640 tgagcagatt ggcccttacc aaggtgtggc tctacggaat caggcttcggtgatgacaag 5700 catatttctc cctagaatgc tgtgccactc actggcttag gagtctcagctctgggtact 5760 ccctctgaat aatgtttgtc cttatctgtg cagagaacac tgtctctaaagcatcctttt 5820 tggcaacgca tttgctcaat caactactga attggtgtta aaattaattttccttttttt 5880 ctcattatgc aaataagaaa ttgagaagca aagctagcag agatttctatcacacctatc 5940 agggatacac aatttccaag aatttcagaa gtgtttggtg ttcctattaacataaatccg 6000 gaaataacac ctgagtgaac tgtcttctaa ttcttcaact ggatggctttttagtgtaaa 6060 agatgttgaa tactgattga ctttttaata attttatagt atatgtcagaaatattgcac 6120 agtccctatt tacatcattc tacagtggtt tttaaaatgt tttaagaataaaaaacatga 6180 aaactttatt tgatttttct gaggaaataa ctttttggat ttaatttcaatgaaaccgtt 6240 gataacattt ccctccccaa caatctctgg caacgatccc tcagattttaatgattatgt 6300 attattacct tttaatacaa gtagaataac actcagggaa tttacaacatttgttatttt 6360 cagtaaatac attggttgaa gtttaaaagt ctatccgtag taaacttacatctttcagga 6420 gcttggtcaa tgtgttctgg acaaagcagg aagatgtgac tgaaatcctgaaaggagccg 6480 gctcctgcag cacaaggata atgatacatc tgggtacatt tctcttcacagcatttgata 6540 gtggctccaa agtgcttaca aaatgcacat tgctgaaagg ggtaaaggagagaaatctct 6600 ttataaaacc ttgaaaagga atatttaaat ataagctggg aaggtataaaaaactctctg 6660 taacatcaca agtaaacaaa ttgaacctgc aaaatattaa acaaaggattcattaaaaat 6720 aataaaatct acattactca atttagtgct ttgtgtgcta ccaactcatccttccattca 6780 aattagaaag ttagaatttc attccttata ttttcaaaaa taaattgtgaagcattttag 6840 aaacaaaacc taaaattttt ttttaaaagc aaatagtaat atggttaaaggggcaggttt 6900 ctatattgag gattattata aagtttttaa atcctaccaa aactagtaataggaacatat 6960 attatttatg agacatatta ctatttttta ccctgcctaa aaataaatacaaataaattc 7020 atcaattata agttaacagg gacacaaatg gttaaagact cacacacaaaaaaaacaaaa 7080 ctacatactt caatgtagca atcaacttca aatttcttaa caaaagatggaaatgttggg 7140 gaaaaaatta gtcatctggt atctttccca tttcaacctg cctccattatcttgcaagtg 7200 gtaaaatgca cagaaataag cctcaaacaa gaggggcagt ctagggcaagtgaacacata 7260 agtcggaaga aattatgtaa aatgttgcat ttacttattc agttttcccttagaatgatt 7320 cacaaactct tcctcattct cccaagtcca ttttgagtat cattttctttgaagagagtc 7380 tgatgggccc tgtactatac agtatgaaat ctctctgtgg gaaatgactatctaacataa 7440 atttttgttt acaccgttac atggtaccta cttgcttatg ccattacatgatcagtttac 7500 ctttttctca acctaatcca agatccttca attgaggcac tatactatctttgtatccaa 7560 agcaccaaaa atgctgcttc aaacaggccc taatagatag gtgttcctatacatatacca 7620 aaaagactta acttttggtg atcttgtttg tgagtgtggc tcataaacagcttagttgag 7680 ataactggag cctcatgtag cagagacagt tggaccctgc taacattactgtggatatct 7740 tcacatgtta ctacattgac tttatattct gctaattaac cagggactacagtagttaaa 7800 attataattg ttttcaatgt tttatgtgta aatctgtatc tcacatactatcaaactctt 7860 cctcactgtc atcagtctac tgcattgaat ccaacataac aaagctaaatgactcctgag 7920 ggctgaatca gaaagaagaa aagaaagaga tacaaaactt tagtcggcccggtggctcac 7980 acctgtaatc ccagcacttt ggaaggccaa ggcgggcgga tcacgaggtcaggagatcga 8040 gaccatcctg gctgatacag tgaaactcca tctctactga aaatacaaaaaattagctgg 8100 acgtggtggt gggcacctgt agtcccagct actcaggagg ctgaagcaggagaagcttct 8160 aaataactca taaacactaa ttactgttgt gacactttaa ttttatacaatatttataag 8220 tatacagaat aacatttcag tgctattttg gcactcaagg gtattaatgcatagcttgag 8280 tattctatag tgtcacctaa atagcttggc gtaatcatgg tcatagctgtttcctgtgtg 8340 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataaagtgtaaagc 8400 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcactgcccgcttt 8460 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcgcggggagagg 8520 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgcgctcggtcgt 8580 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttatccacagaatc 8640 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggccaggaaccgtaa 8700 aaaggccgcg ttgctggcgt ttttcgatag gctccgcccc cctgacgagcatcacaaaaa 8760 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagataccaggcgtttcc 8820 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccggatacctgtc 8880 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtaggtatctcag 8940 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccgttcagcccga 9000 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagacacgacttatc 9060 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtaggcggtgctac 9120 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtatttggtatctg 9180 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgatccggcaaaca 9240 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgcgcagaaaaaa 9300 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagtggaacgaaaa 9360 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacctagatcctttt 9420 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaacttggtctgacag 9480 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttcgttcatccat 9540 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttaccatctggccc 9600 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttatcagcaataaa 9660 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccgcctccatcca 9720 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaatagtttgcgcaa 9780 cgttgttggc attgctacag gcatcgtggt gtcacgctcg tcgtttggtatggcttcatt 9840 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgtgcaaaaaagc 9900 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcagtgttatcact 9960 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaagatgcttttc 10020 tgtgactggt gagtactcaa ccaagtcatt ctgagaatac cgcgcccggcgaccgagttg 10080 ctcttgcccg gcgtcaatac gggataatag tgtatgacat agcagaactttaaaagtgct 10140 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgctgttgagatc 10200 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatcttttactttcaccag 10260 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaataagggcgac 10320 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagcatttatcaggg 10380 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaacaaataggggt 10440 tccgcgcaca tttccccgaa aagtgccacc tgtatgcggt gtgaaataccgcacagatgc 10500 gtaaggagaa aataccgcat caggcgacgc gccctgtagc ggcgcattaagcgcggcggg 10560 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgcccgctccttt 10620 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaagctctaaatcg 10680 ggggctccct ttagggttcc gatttagagc tttacggcac ctcgaccgcaaaaaacttga 10740 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttcgccctttgac 10800 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaacactcaaccc 10860 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcctattggttaaa 10920 aaatgagctg atttaacaaa tatttaacgc gaattttaac aaaatattaacgtttacaat 10980 ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcgggcctcttcg 11040 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttgggtaacgcca 11100 gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaatacgactcacta 11160 ta 11162 2 30 DNA Artificial Sequence TPCF primerdirected to Homo sapiens TNK-TPA gene. 2 atgcatccta ggggaggtcgctgagtagtg 30 3 29 DNA Artificial Sequence TPCR primer directed to Homosapiens TNK-TPA gene. 3 tgcatgccta ggtaccccct agagcccag 29

1. A cell lines that express human mutated tissue-type plasminogenactivator (TNK-TPA), its Collection NO. is CCTCC C200006.
 2. Aconstruction strategy of the cell lines said in claim 1, includes thefollowing steps: (1) Using a DNA sequence from the short arms of humanD,G group chromosomes, or its homologous sequence as leading sequencefor gene targeting to construct a human source gene vector, in which noimportant physiological function gene is found. (2)The target geneTNK-TPA is ligated into the human source vector above with routinemethod, and the recombinant of the vector and TNK-TPA is obtained.(3)TNK-TPA gene is targeted to nucleolus organizing region of human D,Ggroup chromosomes in host cell HT1080 by the above recombinant and thecell line attained by screening.
 3. The construction strategy of thecell lines according to claim 2 wherein the vector has the DNA sequenceas shown in the SEQ No.
 1. 4. The method of preparing mutated humantissue-type plasminogen activator is that the cell lines mentioned inclaim 1 are used as engineering cell line to express and manufactureTNK-TPA protein.